ABSTRACT Although potent antiretroviral therapy (ART) suppresses HIV infection to undetectable levels of plasma viremia, the stably integrated HIV genome continues to persist as a viral reservoir in long lasting resting CD4+ T cells (rCD4). There is substantial ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack a universal measure to assess whether or not a decrease in the latent reservoir has been achieved. The quantitative viral outgrowth assay (Q-VOA) which quantifies inducible, replication-competent HIV-1 from rCD4+ T cells, is used extensively to quantify size of latent viral reservoir, but it provides only a minimal definitive estimate of the size of the latent HIV-1 replication- competent pool. Additionally, the Q-VOA cannot be used to measure the size of the latent replication competent pool HIV-1 in tissues, which represents a major sanctuary site for latent HIV-1.Therefore, there is an urgent need to develop a high throughput, sensitive, and well-validated assay that can quantify replication competent HIV-1 in blood and tissues which will help in developing strategies to eradicate HIV-1. We have recently published a reporter cell-based assay, termed TZA, to quantify inducible replication-competent latent HIV-1. TZA has several advantages over existing technologies in that it (i) is more sensitive; (ii) requires only a small blood volume and (iii) is significantly faster and less labor intensive. Using this assay we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on ART is approximately 70-fold larger than previous estimates. The primary goal of this application is to further develop and characterize this assay so that it can be universally used to quantify the pool of replication-competent HIV-1 in blood and tissues. The Specific Aims of the project are: 1) To characterize the inducible replication-competent latent HIV-1 reservoir in the blood of infected subjects using the TZA. To achieve this goal, we will validate the TZA in the HIV Reservoir Assay Validation and Evaluation Network (RAVEN) project by comparing its sensitivity, dynamic range, and reproducibility with other assays for quantifying replication competent HIV-1 in PBMC from a large and well characterized repository of samples from HIV-infected participants on suppressive ART therapy. As a part of the additional validation of the TZA we will measure the dynamics of the size of replication-competent pool of virus in these cells in subjects who initiated therapy during the acute or chronic stages of infection in subjects from the MACS; 2) To quantify replication-competent HIV-1 in semen and gut associated lymphoid tissue (GALT) from aviremic subjects under ART using the TZA. Ultimately, we anticipate that further development, refinement and validation of TZA will provide a much-needed tool to quantify viral reservoir in blood and tissues that could be widely used by investigators involved in HIV-1 cure studies.